Rat Inner Medullary Collecting Ducts Research Articles

AMPK Stimulates Osmotic Water Permeability in Rat IMCDs

Our previous studies showed that adenosine monophosphate activated kinase (AMPK) is a possible alternate, vasopressin‐independent, regulatory pathway for urea and water transport. Mice lacking a vasopressin receptor (V2R‐KO) are polyuric. Intraperitoneal (IP) injection of metformin to stimulate AMPK significantly increased urine osmolality from 196 ± 32 (control) to 399 ± 52 mOsM at 1h, and 586 ± 41 mOsM at 5h post treatment in the V2R‐KO mice. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we showed that metformin and AICAR, two AMPK activators, significantly increased urea permeability (control:metformin 16 ± 2 to 22 ± 4 ×10−5 cm/s; control:AICAR 19 ± 2 to 23 ± 3 ×10−5 cm/s). In this study, we investigated the role of AMPK in the regulation of water transport in IMCDs. In perfused rat IMCDs, metformin (400 μM) significantly increased osmotic water permeability from 72 ± 42 to 104 ± 61 μm/s. Immunohistochemistry showed that injecting rats IP with metformin increased the membrane accumulation of total aquaporin‐2 (AQP2) and Serine 256 phosphorylated AQP2 (pSer256‐AQP2). Phosphorylation of Serine 256 is required for AQP2 membrane accumulation. In contrast, western blot analysis of biotinylated proteins showed that metformin did not increase urea transporter UT‐A1 membrane accumulation. We conclude that AMPK activation increases: 1) water permeability by increasing the membrane accumulation of total and pSer256‐AQP2; and 2) urea permeability by stimulating the UT‐A1 transporter that is already present in the membrane. These data show that AMPK activation increases water and urea permeability in rat IMCDs. Thus, activation of AMPK may be a novel therapeutic approach to treating nephrogenic diabetes insipidus due to defective vasopressin signaling.Support or Funding InformationNIDDK

Protein kinase C-α mediates hypertonicity-stimulated increase in urea transporter phosphorylation in the inner medullary collecting duct

The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [(32)P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.

Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-type UT-A1 and one expressing a mutated form of UT-A1, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. The phospho-S486-UT-A1 antibody identified UT-A1 protein in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospho-S486-UT-A1 (measured using the phospho-S486 antibody) and of total UT-A1 phosphorylation (measured by (32)P incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-A1 in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-A1 appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-A1 and an S486-UT-A1 phospho-specific antibody. We confirmed that vasopressin increases UT-A1 accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-A1 at S486 in rat IMCDs and that the S486-phospho-UT-A1 form is primarily detected in the apical plasma membrane.

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK-ERK pathway.

Epac Regulation of Urea Transport and the UT‐A1 Urea Transporter in Rat Inner Medullary Collecting Duct.

Urea is critical to the urinary concentrating mechanism and to the regulation of water balance. Vasopressin, through cAMP, stimulates urea transport across perfused rat terminal inner medullary collecting ducts (IMCD) by increasing UT‐A1 phosphorylation and membrane accumulation. Cyclic AMP is known to act through protein kinase A (PKA); however, it can also activate Epac (exchange protein activated by cAMP). We tested whether Epac is involved in regulation of urea transport and UT‐A1 transporter activity in rat IMCD. Functional analysis showed that the Epac activator, Sp‐8‐pCPT‐2 O‐Me‐cAMPS, significantly increased urea permeability in isolated, perfused rat terminal IMCDs by 29%. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT‐A1 in the plasma membrane (biotinylated UT‐A1) and UT‐A1 phosphorylation. To further explore the signaling pathway, we tested whether forskolin could stimulate ERK 1/2 phosphorylation. Forskolin increased ERK 1/2 phosphorylation 25%; this increase was not inhibited by a PKA inhibitor, H‐89, indicating that ERK 1/2 phosphorylation was through Epac. The MEK 1/2 inhibitor, U0126, inhibited forskolin‐stimulated UT‐A1 phosphorylation. We conclude that activation of Epac increases urea transport, UT‐A1 phosphorylation, and UT‐A1 plasma membrane accumulation. The increase in UT‐A1 phosphorylation is mediated by the MEK‐ERK pathway.

Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts

It is well known that Glucagon (Gl) is released after a high protein diet and participates in water excretion by the kidney, principally after a protein meal. To study this effect in in vitro perfused inner medullary collecting ducts (IMCD), the osmotic water permeability (Pf; mum/s) at 37 degrees C and pH 7.4 in normal rat IMCDs (n = 36) perfused with Ringer/HCO(3) was determined. Gl (10(-7) M) in absence of Vasopressin (AVP) enhanced the Pf from 4.38 +/- 1.40 to 11.16 +/- 1.44 microm/s (P < 0.01). Adding 10(-8), 10(-7), and 10(-6) M Gl, the Pf responded in a dose-dependent manner. The protein kinase A inhibitor H8 blocked the Gl effect. The specific Gl inhibitor, des-His(1)-[Glu(9)] glucagon (10(-7) M), blocked the Gl-stimulated Pf but not the AVP-stimulated Pf. There occurred a partial additional effect between Gl and AVP. The cAMP level was enhanced from the control 1.24 +/- 0.39 to 59.70 +/- 15.18 fm/mg prot after Gl 10(-7) M in an IMCD cell suspension. The immunoblotting studies indicated an increase in AQP2 protein abundance of 27% (cont 100.0 +/- 3.9 vs. Gl 127.53; P = 0.0035) in membrane fractions extracted from IMCD tubule suspension, incubated with 10(-6) M Gl. Our data showed that 1) Gl increased water absorption in a dose-dependent manner; 2) the anti-Gl blocked the action of Gl but not the action of AVP; 3) Gl stimulated the cAMP generation; 4) Gl increased the AQP2 water channel protein expression, leading us to conclude that Gl controls water absorption by utilizing a Gl receptor, rather than a AVP receptor, increasing the AQP2 protein expression.

PKC stimulated by glucagon decreases UT-A1 urea transporter expression in rat IMCD

It is well-known that glucagon increases fractional excretion of urea in rats after a protein intravenous infusion. This effect was investigated by using: (a) in vitro microperfusion technique to measure [(14)C]-urea permeability (Pu x 10(-5)cm/s) in inner medullary collecting ducts (IMCD) from normal rats in the presence of 10(-7)M of glucagon and in the absence of vasopressin and (b) immunoblot techniques to determine urea transporter expression in tubule suspension incubated with the same glucagon concentration. Seven groups of IMCDs (n = 47) were studied. Our results revealed that: (a) glucagon decreased urea reabsorption dose-dependently; (b) the glucagon antagonist des-His(1)-[Glu(9)], blocked the glucagon action but not vasopressin action; (c) the phorbol myristate acetate, decreased urea reabsorption but (d) staurosporin, restored its effect; e) staurosporin decreased glucagon action, and finally, (f) glucagon decreased UT-A1 expression. We can conclude that glucagon reduces UT-A1 expression via a glucagon receptor by stimulating PKC.

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct

Collecting duct cells swell when exposed to arginine vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated, perfused rat inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomyosin). When a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradient and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl cotransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs from NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath caused significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry.

Vasopressin Increases Plasma Membrane Accumulation of Urea Transporter UT-A1 in Rat Inner Medullary Collecting Ducts

Urea transport, mediated by the urea transporter A1 (UT-A1) and/or UT-A3, is important for the production of concentrated urine. Vasopressin rapidly increases urea transport in rat terminal inner medullary collecting ducts (IMCD). A previous study showed that one mechanism for rapid regulation of urea transport is a vasopressin-induced increase in UT-A1 phosphorylation. This study tests whether vasopressin or directly activating adenylyl cyclase with forskolin also increases UT-A1 accumulation in the plasma membrane of rat IMCD. Inner medullas were harvested from rats 45 min after injection with vasopressin or vehicle. UT-A1 abundance in the plasma membrane was significantly increased in the membrane fraction after differential centrifugation and in the biotinylated protein population. Vasopressin and forskolin each increased the amount of biotinylated UT-A1 in rat IMCD suspensions that were treated ex vivo. The observed changes in the plasma membrane are specific, as the amount of biotinylated UT-A1 but not the calcium-sensing receptor was increased by forskolin. Next, whether forskolin or the V(2)-selective agonist dDAVP would increase apical membrane expression of UT-A1 in MDCK cells that were stably transfected with UT-A1 (UT-A1-MDCK cells) was tested. Forskolin and dDAVP significantly increased UT-A1 abundance in the apical membrane in UT-A1-MDCK cells. It is concluded that vasopressin and forskolin increase UT-A1 accumulation in the plasma membrane in rat IMCD and in the apical plasma membrane of UT-A1-MDCK cells. These findings suggest that vasopressin regulates urea transport by increasing UT-A1 accumulation in the plasma membrane and/or UT-A1 phosphorylation.

Mitochondrial Expression of Arginase II in Male and Female Rat Inner Medullary Collecting Ducts

Microdissected rat proximal straight tubules (PST) and inner medullary collecting ducts (IMCD) highly produce urea from l-arginine, supporting the expression of the mitochondrial arginase II. However, IMCD contain a very low density of mitochondria compared with PST. Recently, arginase II has been localized by immunohistochemistry in rat PST but not IMCD. This study was designed to verify whether rat IMCD express arginase II and to identify its subcellular localization. We developed an antibody raised against arginase II that allowed the detection of a band of 38 kDa corresponding to arginase II on immunoblots. In male and female rat kidneys, Western blot analyses revealed that arginase II was highly expressed in the inner medulla (IM), the outer stripe of the outer medulla (osOM), and the deep cortex. Immunocytochemistry demonstrated that arginase II was homogeneously expressed in IMCD. Proteins of the cytosolic and mitochondrial fractions extracted from osOM and IM and analyzed by Western blot showed that 86% of arginase II was associated with mitochondria. The molecular weight of arginase II was similar in the cytosolic and mitochondrial fractions. Immunoelectron microscopy confirmed the presence of arginase II in the mitochondria of IMCD. In conclusion, arginase II is expressed in mitochondria of male and female rat IMCD.

Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA.

The UT-A1 urea transporter plays an important role in maintaining the hyperosmolar milieu of the inner medulla. Vasopressin increases urea permeability in rat terminal inner medullary collecting ducts (IMCDs) within 5-10 min. To elucidate the mechanism, IMCD suspensions were radiolabeled with [(32)P]orthophosphate. UT-A1 was immunoprecipitated and analyzed by autoradiogram and Western blot. Both the 97- and 117-kDa UT-A1 proteins were phosphorylated. Vasopressin treatment increased the phosphorylation of both UT-A1 proteins at 2 min, which peaked at 5-10 min and remained elevated for up to 30 min. There was a discernable increase in UT-A1 phosphorylation with 10 pM and a 50% increase with 10-100 nM vasopressin. 1-Desamino-8-D-arginine vasopressin (dDAVP) or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) also increased UT-A1 phosphorylation. The vasopressin-stimulated increase in UT-A1 phosphorylation was blocked by H-89 or a specific peptide inhibitor of protein kinase A. Phosphatase inhibitors (okadaic acid, calyculin) increased UT-A1 phosphorylation. We conclude that vasopressin increases UT-A1 phosphorylation via protein kinase A within 2-5 min in rat IMCDs. This suggests that phosphorylation of UT-A1 may be the mechanism by which vasopressin rapidly increases urea permeability in vivo.

Maturation of aldose reductase expression in the neonatal rat inner medulla.

Newborns are less able to concentrate urine than adults are. With development of the concentrating system and a hypertonic medullary interstitium, there is a need to generate intracellular osmolytes such as sorbitol, which is produced in a reaction catalyzed by the enzyme aldose reductase. We sought to discriminate between two possible mechanisms of aldose reductase induction during development: (a) a response to an osmotic stimulus generated by the concentrating mechanism; or (b) part of the genetic program for development of the kidney. We measured the change in aldose reductase mRNA and activity in terminal inner medullary collecting ducts (IMCDs) microdissected from Sprague-Dawley rats during the first month of life. Aldose reductase mRNA was assayed by Northern analysis of total RNA from inner medulla and by detection of the reverse transcription-polymerase chain reaction (RT-PCR) product obtained from single IMCDs using aldose reductase-specific primers. Aldose reductase activity was measured in IMCDs taken from the same rats using a fluorescent microassay. Newborn rat IMCDs had minimal aldose reductase mRNA or activity, however mRNA was readily detected in IMCDs from rats older than 3 d of age, with peak expression occurring at 1-3 wk of age before decreasing to adult levels. In contrast, the mRNA level for a housekeeping metabolic enzyme, malate dehydrogenase, did not change during maturation. Aldose reductase enzyme activity was readily detectable by 6 d of age, peaked at 20 d, then decreased to adult levels. Urine osmolality remained < 600 mosmol/kg until 16 d, then increased to > 1,100 mosmol/kg after 20 d. Thus, aldose reductase mRNA and activity increased before urinary osmolality reached 870 mosmol/kg. Because urine osmolality may not be indicative of inner medullary osmolality and because mother's milk may provide excessive free water to the pups under 3 wk of age, half of the animals in several litters were separated from their mothers for 1 d and inner medullary osmolality, in addition to urine osmolality, was measured by vapor pressure osmometry, while aldose reductase mRNA was assessed densitometrically in IMCDs after RT-PCR. Although fluid restriction resulted in a near doubling of urine osmolality and a tendency towards increased aldose reductase mRNA, there was no consistently significant increase in aldose reductase mRNA or inner medullary osmolality during the first 13 d of life compared to the suckling animals. On the other hand, 2-3-wk-old rats showed significant increases in aldose reductase mRNA, accompanied by increases in inner medullary osmolality, after fluid restriction. Thus, the dissociation between the increases in aldose reductase expression and inner medullary hyperosmolality indicates that the maturational induction of the aldose reductase gene is not a consequence of osmotic stimulation, but rather, part of the developmental program of the kidney.

Endothelin-1 inhibits AVP-stimulated osmotic water permeability in rat inner medullary collecting duct.

Endothelin causes diuresis despite an accompanying decrease in glomerular filtration rate and renal plasma flow. Binding sites for endothelin are located not only in glomeruli but also in the inner medulla, possibly in inner medullary collecting ducts (IMCD). To determine whether endothelin has a direct tubular effect, effects of endothelin on water and urea transport were investigated using isolated microperfusion of rat IMCD segments in vitro. Endothelin, at 10(-10) and 10(-8) M, reversibly inhibited 10(-11) M arginine vasopressin (AVP)-stimulated osmotic water permeability (Pf) by 18 and 24%, respectively. Endothelin (10(-8) M) also inhibited Pf by 23% in the presence of a much higher dose of AVP (10(-9) M), whereas endothelin had no effect on Pf in the absence of AVP. On the other hand, 10(-8) M endothelin did not inhibit Pf stimulated by 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). Endothelin had no inhibitory effect on AVP-stimulated urea permeability. These data suggest that endothelin can cause diuresis by inhibiting AVP-stimulated Pf in IMCD and that the site of action is previous to cAMP generation.

Urea gradient-associated fluid absorption with sigma urea = 1 in rat terminal collecting duct

It has been proposed that inner medullary collecting ducts (IMCDs) can absorb fluid in the absence of a transepithelial osmolality gradient if a perfusate-to-bath urea gradient is present. Such a process has been suggested to be caused by a nonunity reflection coefficient for urea (sigma urea less than 1). However, our recent measurements of sigma urea yielded values not significantly different from 1.0. The present study was done to readdress the possibility of direct coupling of water and urea transport in the rat IMCD. Isolated rat terminal IMCD segments were studied in the presence of 10(-10) M vasopressin with the osmolality of the perfusate equal to that of the peritubular bath but with a perfusate-to-bath urea gradient (bath osmolality balanced with NaCl). We measured both fluid absorption rate and urea concentration in collected fluid and calculated the osmolality of the collected fluid. We observed rapid fluid absorption associated with substantial urea absorption. The urea absorption caused a large fall in the osmolality of the collected fluid with respect to the bath. Simulations with a mathematical model of an isolated perfused tubule revealed that the transepithelial osmolality gradient generated along the length of tubule (caused by urea absorption) was large enough to account for the fluid absorption. Measurement of sigma urea with the "zero-flux" (or null point) method revealed a value of 1.00 +/- 0.02. Thus we conclude that the observed fluid absorption is the result of a transepithelial osmolality gradient generated by rapid urea absorption and does not require sigma urea less than one.

Effect of vasopressin on sodium transport across inner medullary collecting duct

We have used rat inner medullary collecting ducts (IMCD) perfused "in vitro" to study the effect of vasopressin (VP) on the unidirectional Na+ flux (in nmol.cm-2.s-1). We found that, at a high perfusion rate in the basal state, 24Na lumen-to-bath flux (Jl----b) was greater than the bath-to-lumen flux (Jb----l) (4.88 +/- 0.15 vs. 2.57 +/- 0.21), resulting in a significant net flux (Jnet) (P less than 0.001). Addition of 10 microU/ml of lysine vasopressin (LVP) to the bath produced a stable increase in Jl----b to 6.66 +/- 0.35 (P less than 0.001) without significant effect on Jb----l. Measuring directly the net flux absorption at lower perfusion rate (8 nl/min), we observed that LVP (10 microU/ml) produced a reversible stimulation on Jnet from 1.39 +/- 0.14 to 2.79 +/- 0.23 (P less than 0.01). The transtubular potential difference (PD) measured in the middle and final third of IMCD showed a small but significant PD (0.30 +/- 0.02 mV lumen positive) that increased significantly to 0.60 +/- 0.04 mV in the presence of LVP. However, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 10(-4) M) added to the bath fluid did not change the JNa+l----b, nor was 1-desamino-8-D-arginine vasopressin (50 microU/ml), a specific V2-agonist, able to increase the Na+. We also demonstrated that JNa+l----b stimulated by LVP from 4.70 +/- 0.08 to 6.33 +/- 0.26 (P less than 0.01) was completely and reversibly inhibited by V1-antagonist, d(CH2)Tyr(Me)AVP, to 4.79 +/- 0.05. On the other hand, the absence of Ca2+ in the bath or the addition of amiloride to the lumen fluid or ouabain to the bath fluid completely inhibited AVP-stimulated JNa+l----b. Therefore, AVP and LVP increase Na+ absorption in the rat IMCD by increasing the Na+ outflux, probably generated by an increase of luminal membrane Na+ permeability modulated by extracellular Ca2+ and mediated through V1-receptors and independent of cAMP cascade.

Effect of furosemide on water and urea transport in cortical and inner medullary collecting duct

The present in vitro microperfusion study examined whether furosemide has an effect on hydraulic conductivity (Lp X 10(-6) cm/sec.atm) and 14C-urea permeability (Pu X 10(-5) cm/sec) in inner medullary collecting ducts (IMCD) and cortical collecting tubules (CCT) isolated from rat and rabbit kidneys. Furosemide added to the bath fluid decreased arginine-vasopressin (AVP)-stimulated Lp of rat IMCD in a dose-dependent manner, with the threshold effect at 10(-6) M. Furosemide (10(-4) M) reduced Lp from 20.5 +/- 2.3 to 12.1 +/- 1.2 (P less than 0.01) reversibility, but had no effect when added to the perfusate. In addition, furosemide reduced dibutyryl cyclic AMP-stimulated Lp from 20.3 +/- 1.1 to 11.2 +/- 1.6 (P less than 0.01). This effect of furosemide was also observed with indomethacin, a PGE2 synthesis inhibitor. The addition of indomethacin (10(-4) M) to AVP (50 microU/ml) increased Lp from 24.7 +/- 2.3 to 29.7 +/- 2.8 (P less than 0.001), which was reduced to 20.3 +/- 2.6 (P less than 0.001) when furosemide was added to indomethacin in the bath. The inhibitory effect of furosemide on AVP-stimulated Lp was also observed in rabbit IMCD (Lp decreased from 12.8 +/- 0.8 to 5.15 +/- 1.46, P less than 0.02), but it was not observed in the CCT isolated from rabbit kidneys (7.96 +/- 1.87 with AVP vs. 7.94 +/- 1.41 with AVP + furosemide). Furthermore, in rat IMCD the stimulatory effect of AVP on Pu from 7.7 +/- 0.4 to 26.8 +/- 1.3 was reduced by furosemide to 19.7 +/- 1.2 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hypertonicity on ADH-stimulated water permeability in rat inner medullary collecting duct

To assess the effects of increased tonicity on water reabsorption (Jv) in inner medullary collecting ducts (IMCD), antidiuretic hormone (ADH)-stimulated Jv and water permeability (PF) were determined in microperfused IMCD dissected from the inner medulla of rat kidney. In IMCD exposed to a 150-mosmol/kgH2O gradient in isotonic bath, ADH-stimulated PF averaged 719 +/- 93 microns/s. Symmetric addition of 75 mM NaCl to perfusate and bath resulted in a significant augmentation of ADH-stimulated PF (56%) that was reversible when initial solutions were restored. Despite the increase in PF, JV did not change but would have decreased by 16% (P less than 0.01) had PF not increased, because of the greater absolute axial increase in luminal tonicity that occurs with more hypertonic luminal solutions. When 150 mM mannitol was used to increase tonicity, similar effects were observed. However, 150 mM urea had no effect on ADH-stimulated PF. In IMCD exposed to 8-para-(chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, addition of 75 mM NaCl to both and perfusate also resulted in a 76% increase in PF. These results are the first to demonstrate directly that increased effective tonicity augments ADH-stimulated PF in rat IMCD at a site distal to adenosine 3',5'-cyclic monophosphate generation. This effect may contribute to maintenance of medullary interstitial tonicity during antidiuresis by ensuring that most water reabsorption occurs more proximally within the IMCD.

Mechanism of diuretic action of U-62,066E, a κ opioid receptor agonist

The mechanism of the diuretic action of U-62,066E, a highly selective κ opioid agonist, was examined in unanesthetized rats and in isolated perfused inner medullary collecting ducts (IMCD). In Long-Evans rats, U-62,066E caused a dose-dependent increase in urine flow and a decrease in urine osmolality without affecting urinary excretion of Na +. The diuretic effect of U-62,066E was blocked by MR-2266, a κ opioid receptor antagonist. U-62,066E showed no diuretic effect in homozygous hereditary diabetes insipidus rats (Brattleboro strain). In water-deprived rats, U-62,066E markedly inhibited plasma arginine vasopressin (AVP) levels through a κ receptor-mediated mechanism. In rat IMCD perfused in vitro, 10 −5 M U-62,066E did not inhibit either the baseline or the AVP-stimulated osmotic water permeability. We conclude that the inhibition of the release of AVP is the major if not the entire mechanism of the diuretic action of U-62,066E in rats. Although we ruled out the effect of this drug on the water permeability of IMCD, possible direct effects on other nephron structures remain to be established.

Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium.

To compare passive urea transport across the inner medullary collecting ducts (IMCDs) and the papillary surface epithelium (PSE) of the kidney, two determinants of passive transport were measured, namely permeability coefficient and surface area. Urea permeability was measured in isolated perfused IMCDs dissected from carefully localized sites along the inner medullas of rats and rabbits. Mean permeability coefficients (X 10(-5) cm/s) in rat IMCDs were: outer third of inner medulla (IMCD1), 1.6 +/- 0.5; middle third (IMCD2), 46.6 +/- 10.5; and inner third (IMCD3), 39.1 +/- 3.6. Mean permeability coefficients in rabbit IMCDs were: IMCD1, 1.2 +/- 0.1; IMCD2, 11.6 +/- 2.8; and IMCD3, 13.1 +/- 1.8. The rabbit PSE was dissected free from the underlying renal inner medulla and was mounted in a specially designed chamber to measure its permeability to urea. The mean value was 1 X 10(-5) cm/s both in the absence and presence of vasopressin (10 nM). Morphometry of renal papillary cross sections revealed that the total surface area of IMCDs exceeds the total area of the PSE by 10-fold in the rat and threefold in the rabbit. We conclude: the IMCD displays axial heterogeneity with respect to urea permeability, with a high permeability only in its distal two-thirds; and because the urea permeability and surface area of the PSE are relatively small, passive transport across it is unlikely to be a major source of urea to the inner medullary interstitium.